WO1991015233A1 - Emploi de protease nexine-i induisant la cicatrisation de plaies - Google Patents

Emploi de protease nexine-i induisant la cicatrisation de plaies Download PDF

Info

Publication number
WO1991015233A1
WO1991015233A1 PCT/US1991/002161 US9102161W WO9115233A1 WO 1991015233 A1 WO1991015233 A1 WO 1991015233A1 US 9102161 W US9102161 W US 9102161W WO 9115233 A1 WO9115233 A1 WO 9115233A1
Authority
WO
WIPO (PCT)
Prior art keywords
wound
protease nexin
pharmaceutical composition
collagen
occlusive
Prior art date
Application number
PCT/US1991/002161
Other languages
English (en)
Inventor
Randy W. Scott
Richard A. Berg
Original Assignee
Invitron Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Invitron Corporation filed Critical Invitron Corporation
Priority to EP91907406A priority Critical patent/EP0556178B1/fr
Priority to DE69129071T priority patent/DE69129071T2/de
Publication of WO1991015233A1 publication Critical patent/WO1991015233A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
    • A61L2300/104—Silver, e.g. silver sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A61L2300/254—Enzymes, proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • A61L2300/406—Antibiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/45—Mixtures of two or more drugs, e.g. synergistic mixtures

Definitions

  • This invention relates generally to the field of wound healing methods and to compositions and dressings useful in carrying out such methods. More specifically, this invention relates to using protease nexin-I (PN-I) to promote wound healing and to compositions and dressing which include (PN-I) and are useful in carrying out the wound healing methodology.
  • PN-I protease nexin-I
  • Wound healing has three distinct phases: (1) inflammation; (2) cell migration and proliferation; and (3) remodeling.
  • the inflammatory phase the wound is described by proteases released by inflammatory cells.
  • Various lymphokines are secreted from neutrophils and macrophages that modulate the next phase of the wound healing.
  • the second phase includes fibroblast migration, proliferation and the synthesis of new extracellular matrix molecules. These events appear to occur in a definite order where extracellular matrix molecules including fibronectin, collagen, and proteoglycans are secreted into the granulation bed.
  • the first phase peaks at 3 days.
  • the second phase of wound healing normally peaks at approximately one to two weeks after injury and is followed by a much longer third phase of tissue remodeling that begins within weeks and may last several months .
  • the remodeling phase the connective tissue matrix matures as the disorganized collagen fibers are replaced by much thicker, more aligned collagen molecules. This tissue remodeling eventually contributes to the tensile strength of the wound and is sometimes accompanied by scar formation.
  • protease nexins ir- reversibly bind to serine proteases at their catalytic sites (Baker, J.B., et al., Cell (1980) 2_l:37-45) and ef ⁇ fect the clearance of the bound proteases via receptor- mediated endocytosis and lysosomal degradation (Low, D.A., et al., Proc Natl Acad Sci (USA) (1981) 78 . :2340-2344; Baker, J.B., et al . , in The Receptors 3 (1985), Conn, P.M., ed, Academic Press, in press) .
  • Protease nexin-I has been purified from serum-free medium conditioned by human foreskin cells (Scott, R.W., et al., J Biol Chem (1983) 5 : 1043910444) .
  • Protease nexin-I is a 43 kD glycoprotein which is released by fibroblasts, myotubes, heart muscle cells, and vascular smooth muscle cells. Its release, along with that of plasminogen activator, is stimulated by phorbol esters and by mitogens (Eaton, D.L., et al. , J Cell Biol (1983)
  • Native PN-I is an approximately 400 amino acid protein containing about 10% carbohydrate. Since it is present only in trace levels in serum, it apparently functions at or near the surfaces of interstitial cells. PN-I inhibits all the known activators of urokinase proenzyme, plasmin, trypsin, thrombin, and factor Xa (Eaton, D.L., et al . , J Biol Chem (1984) 259:6241) . It also inhibits tissue plasminogen activator and urokinase.
  • NPF neurite-promoting factor
  • Protease nexin-I is a serine protease inhibitor member of the serpin super family which is synthesized and secreted by cultured human fibroblasts.
  • the protein represents about 1% of the secreted proteins of fibroblast and has a molecular weight of 43 kD. It reacts rapidly with trypsin, thro bin, urokinase and plasmin to inhibit these serine proteases . It does not react with chymo- trypsin like proteases or PMN elastase.
  • the protein has a high affinity for heparin and heparan-sulfate and can be readily purified by heparin affinity chromatography.
  • protease nexin has been shown to be identical with glia derived neurite promoting factor and is 30% homologous with antithrombin III.
  • thrombin The mitogenic activity of thrombin is effective only when added to cultures at concentrations above the concentrations of secreted PN-I (Baker, J.B., et al . , J Cell Physiol (1982) 112:291; Low, D.A., et al . , Nature (1982) 298: 2476) .
  • Thrombin is also known to cleave and inactivate acidic fibroblast growth factor Lobb, R.R., Biochemistry 2_7_ : 2572-2578.
  • PN- I has an antiinflammatory function, since PN-I secretion by synovial fibroblasts increases dramatically when the cells are treated with interleukin-I (Krane, S., Arth Rheum (19 ) 2_7_:S24). PN-I may also have a neurological function, since the above-mentioned identical protease inhibitor stimulates neurite extension (Monard et al., Prog Brain Res (1983) 58:359) .
  • protease nexin has been reported to have several unique biological properties including the promotion of neural outgrowth in addition to endothelial cell migration and in addition of the inhibition of matrix destruction by fibrosarcomas. The specific role of serine proteases in these processes is not known. However, protease nexin was found to have an effect on the extracellular matrix of fibroblasts in culture. In connection with the conception of the present invention, we hypothesized that the use of protease nexin in several systems might help to elucidate roles of serine proteases in several cellular processes including cell migration, differentiation, and tissue remodeling. In order to obtain the present invention, we tested our hypothesis that protease nexin may play a role in promoting wound healing.
  • PN-I protease nexin-I
  • Pharmaceutical compositions comprised of carriers having PN-I dispersed uniformly therein are disclosed as are such compositions further comprising antibiotics.
  • a variety of wound dressings are taught which are comprised of a sup ⁇ port surface having an absorbent material thereon sur- rounded by a pressure-sensitive adhesive strip. In some cases, the wound bed is kept moist with an occlusive dressing.
  • the PN-I is present on the absorbent material, by itself, or in combination with other excipients or active components such as antibiotics or other macro molecules such as collagen, proteoglycan or heparin.
  • a primary object of the invention is to provide a method for increasing the rate of wound healing by the application of PN-I.
  • Another important object is to provide a pharmaceutical composition for topical application which is comprised of PN-I dispersed uniformly throughout a car ⁇ rier with acceptable pharmacological properties .
  • Another object is to use PN-I in combination with other wound healing factors such as fibroblast growth factor, transforming growth factor beta or other bioactive molecules which aid in wound healing.
  • Yet another important object is to provide a wound dressing comprised of a support base having PN-I thereon.
  • An advantage of the present invention is that the method of applying PN-I to a wound facilitates the return of normal tissues.
  • a feature of the present invention is that the PN-I can be incorporated into a wide variety of different topical pharmaceutical compositions or placed on a variety of wound dressings to obtain the desired results of enhancing wound healing.
  • Another object is to prevent the degradation of other wound healing agents such as growth factors, collagen, fibronectin and other extracellular matrix molecules so as to promote healing.
  • Another object is to lengthen the lifespan of collagen implants for cosmetic and therapeutic treatments. Another advantage results from the cotreatment of wounds with PN-I and growth factors wherein PN-I extends the half-life of the growth factors in the wound bed by preventing proteolytic degradation.
  • Figure 1 shows the nucleotide sequence of the coding region of PN-18 and the deduced amino acid sequence of PN-I alpha.
  • Figure 2 shows the nucleotide sequence of the coding region of PN-33 and the deduced amino acid sequence of PN-I beta.
  • Figure 3 is a graph showing the hydroxyproline content per dry weight of skin in a full thickness wound.
  • Figure 4 is a graph showing the hydroxyproline content per DNA in a full thickness wound.
  • Figure 5 is a graph showing the modulus of the stress strain curves in a full thickness wound.
  • protease nexin-I and protease nexin shall be synonymous and shall refer to a protein which is active in the standard diagnostic assays for PN-I, which are based on four criteria, as follows: (1) The protein complexes to thrombin; (2) this complexation is accelerated by heparin; (3) the protein complexed to thrombin binds to fibroblasts; and (4) heparin inhibits this binding.
  • PN-I is distinguishable from the two other protease nexin factors, PN-II and PN- III (Knauer, D.J., et al. , J Biol Chem (1982) 257:15098- 15104) .
  • Recombinant PN-I was purified to homogeneity from serum-free medium conditioned by Chinese hamster ovary cells.
  • Recombinant cells for producing PN-I are cultured under conditions suitable for the host in question, and the protein is recovered from the cellular lysate or from the medium, as determined by mode of expression. Purification of the protein can be achieved using methods similar to that disclosed by Scott, R.W. , et al., J Biol Chem (1985) 260:7029-7034, incorporated herein by reference.
  • the purified material shows the properties exhibited by PN-I when contained in conditioned medium, including formation of sodium dodecylsulfate-stable complexes with thrombin, urokinase, and plasmin; inhibition of protease activity; and heparin-enhanced inhibition of thrombin.
  • N-terminal amino acid sequence of the isolated, purified protease nexin was determined for the first 34 amino acids to be: Ser-His- Phe-Asn-Pro-Leu-Ser-Leu-Glu-Glu-Leu-Gly-Ser-Asn-Thr-Gly- Ile-Gln-Val-Phe-Asn-Gln-Ile-Val-Lys-Ser-Arg-Pro-His-Asp- Asn-Ile-Val-Ile.
  • compositions or “formulations” of the invention refer to any mixture containing the purified PN-I which protein is formulated according to its pharmaceutical applications for wound healing.
  • the protein is formulated into compositions using pharmaceutically acceptable excipients, as is understood by practitioners of the art, and disclosed, for example, in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton, PA.
  • PN-I alpha and PN-I beta There are two forms of PN-I, PN-I alpha and PN-I beta. They are highly homologous and contain 378 and 379 amino acids, respectively in the mature sequence, differing only at position 310 where the Arg of PN-I alpha is replaced by Thr-Gly in PN-I beta. Both have a 19 amino acid signal beginning at Met. The location of the N- terminus is deduced from the sequenced native protein and it is highly likely this is correct; however, there is a small probability that alternate processing site(s) may also be utilized.
  • purified or pure refers to material which is free from substances which normally accompany it as found in its native state.
  • pure PN-I-encoding DNA refers to DNA which is found in isolation from its native environment and free of association with DNAs encoding other proteins normally produced by cells natively produc ⁇ ing PN-I.
  • “Pure” PN-I refers to PN-I which does not contain materials normally associated with its in situ environment in human or other mammalian tissue. Of course, “pure” PN-I may include materials in covalent as ⁇ sociation with it, such as glycoside residues or materials introduced, for example, for formulation as a therapeutic. "Pure” simply designates a situation wherein the substance referred to is, or has been, isolated from its native environment and materials which normally accompany it.
  • PN-I is somewhat expensive and need not be applied in a highly concentrated form, it is generally formulated in a pharmaceutical composition by dispersing PN-I in a carrier. PN-I is also readily bound to extracellular matrix molecules, one may be applied as a dilute solution to the wound bed followed by allowing the liquid to absorb into the wound bed.
  • PN-I is not applied in a pure form to a wound, but is formulated in combination with one or more excipients and/or other active ingredients such as collagen, proteoglycan, heparin, heparin sulfate, gelatin, hyaluronic acid, la in, fibronectin or fibrin.
  • the PN-I is generally present in the formulation as a minor ingredient i.e., less than 50% and more generally about 0.01% to 1% PN-I by weight and more preferably about 0.05 to 0.5% by weight PN-I.
  • the amount of the PN-I needed in order to obtain an improvement in the rate of wound healing varies depending on the particular type of wound and the particular individual. Further, the number of applications and the period of time over which the applications are made can vary considerably depending on factors such as the type of wound, size of wound, age and size of the patient and general health of the patient. The precise amounts, numbers and periods of administration can be determined by the health care provider.
  • a composition comprised of 0.01 to 1.0 weight percent of PN-I and 99.99 to 99.0 weight percent of excipient can be applied to a wound on a daily basis over a period of one month. The composition is applied in an amount of about 0.01 to 20 grams per square centimeter of wound surface and the wound should be cleaned as needed.
  • the protease nexin-I of the invention is preferably formulated in a semisolid cream or ointment like or more preferably gel formulation.
  • the formulation may be in the form of a solution having the protease nexin-I therein.
  • the protease nexin-I solution may be applied to a full thickness wound after the wound is cleaned and the surface of the wound is allowed to dry slightly after which there is applied about 0.1-lOOug per square centimeter of wound.
  • the type of formulation and amount of protease nexin-I applied will be determined to a large extent by the care giver treating the wound.
  • protease-nexin I may be applied per square centimeter of wound.
  • protease nexin-I it may be necessary for the protease nexin-I to be applied periodically, such as every day, or every other day depending on the individual, the type of wound and the stage of the wound healing.
  • the amount of protease nexin-I and the frequency at which it is applied is a matter which can readily determined by the care giver.
  • a protease nexin-I solution was applied in an amount of about 5-45mg per 4 square centimeters of wound and improved wound healing results were observed as compared with a wound not treated with protease nexin-I.
  • different tests were carried out as described further below. However, it is important to note that one important test involves the determination of the hydroxyproline content of the wound as it continues to heal. The hydroxyproline content is an estimation of the collagen content of the wound.
  • a preferred embodiment of the invention involves dispersing PN-I in collagen or a collagen-based dressing which is then applied to a wound surface.
  • the collagen is present as the excipient in amount ranges as indicated above.
  • the PN-I could be used in a higher concentration with collagen, e.g., 1.0-10.0% PN-I with 99.0 to 90.0% collagen.
  • a preferred embodiment of the present invention involves the use of collagen material.
  • the collagen is obtained from a natural source.
  • the collagen used in connection with the present invention is not “native” or “natural” collagen. It has been modified to some extent in order to purify the collagen material and change its structure in an attempt to eliminate the generation of an immune response when the collagen comes into contact with living tissue.
  • Such collagen is generally referred to as "pharmaceutical grade collagen”.
  • pharmaceutical grade collagen A general description of collagen and how "native” or “natural” collagen is modified to obtain a pharmaceutical grade collagen is put forth below.
  • Native collagen consists in large part of a triple helical structure containing repeating triplet sequences composed of glycine linked to two additional amino acids, commonly proline and hydroxyproline; thus, glycine appears in every third position in the chain.
  • all collagen chains contain regions at each end which do not have the triplet glycine sequence and are thus not helical. These regions are thought to be responsible for the immunogenicity associated with most collagen preparations. Immunogenicity can, in large part, be mitigated by removal of these regions to produce "atelopeptide" collagen. This can be accomplished by digestion with proteolytic enzymes such as trypsin, chymotrypsin, papain, or pepsin.
  • telopeptide regions are also responsible for forming the cross-links which aid in stability of the fibrillar structure. These regions contain aldehydes capable of cross-linkage to lysine residues. Atelopeptide collagen must be cross-linked artificially, if it is so desired.
  • Type I which is present in skin, tendon, and bone, and which is made by fibroblasts
  • Type III which is found primarily in skin and is associated with Type I.
  • Other types reside in specialized membranes, cartilage, or at cell surfaces.
  • Types I and III contain similar numbers of amino acids in their helices; however, Type III (but not Type I) contains two adjacent cysteines at the C- terminal ends of the triple helix which are capable of forming interchain cross-links.
  • Type I collagen contains one alpha 2 (I) and two alpha 1 (I) chains each of which contains 1014 amino acids in its triplet region; there are several carbohydrate moieties present on each chain.
  • Type III collagen contains only alpha 1 (III) (3 chains) which contain 1026 residues in their triplet regions. As stated above, the presence in Type III of a pair of adjacent cysteine residues at the carboxy terminal end of the triplet region results in stability of the interchain cross-links. Both collagens contain short nontriplet ends (telopeptides) .
  • the reconstituted fibrillar atelopeptide skin collagen used in this invention contains the atelopeptide forms of both Type I and Type III; the bone collagen powder consists of the atelopeptide form of Type I exclusively.
  • a pharmaceutical grade collagen material sold under the tradename "Zyderm" can be used in con ⁇ nection with this invention.
  • PN-I Recombinant PN-I was tested on guinea pig full thickness accession wound model. Guinea pigs were given two 2 x 2 centimeter wounds on the sides of their backs equidistant from the midline. The wounds were bandaged for 24 hours using a Bioclusive dressing. At 24 hours, the guinea pigs were given a dose of 5.25 or 45 micrograms PN-I in a volume of 300 microliters onto one wound bed. The second wound on the same animal served as control .
  • the guinea pigs were allowed to heal for either six or 12 days after which time the wound was evaluated by determining the hydroxyproline per dry weight, the hydroxyproline per DNA and the tensile-strength of the excised wounds. The results of these evaluations are shown respectively in Figures 3, 4 and 5.
  • the wound beds were removed, fixed in Bouin's fixative, embedded in paraffin sections at 5 u , and stained with Mason's Trichrome stain for histological examination. Comparing the experimental and control wounds, the results indicated that in six days the hydroxyproline per gram dry weight was increased significantly from 9.42 +2.3 to 13.13 +1.6 mg per gram. Hydroxyproline per DNA increased from 3.14 +.88 to 4.6 +.62 micrograms per microgram; however, the modulus of the stress-strain curves was not different.
  • Example 1 We chose to use the full thickness dermal wound model in guinea pigs because this model provides a relatively large granulation bed in which the early phases of wound healing can be studied and quantified.
  • mice Female Hartley Albino guinea pigs weighing 400 grams were obtained from Hilltop Breeders, maintained in a vivarium and fed guinea pig chow and water ad. lib. for one week to insure that they were healthy. On the day prior to surgery, the backs of the guinea pigs were shaved and depilated. On the day of surgery, two full thickness 2 x 2 centimeter wounds were prepared on either side of the middle of the back. The normal skin, excised to the level of the panniculus carnosus, was reserved for data analysis.
  • the wounds were lavaged with 0.3 ml penicillin/ streptomycin, Gibco Co., at a concentration of 100 units per ml and bandaged with "Bioclusive" transparent dress ⁇ ing. Twenty-four hours after surgery, the wound beds were uninfected and dry.
  • the surface of the "Bioclusive" dressing was swabbed with an alcohol wipe, 0.3 ml sterile solution of 20 mM sodium citrate, 300 mM sodium chloride buffer pH 7.2 was injected through the dressing onto the control wound site.
  • the experimental site received 0.3 ml of sterile buffer containing a dose of protease nexin.
  • the dressing was removed, the wound bed treated with a triple antibiotic ointment containing Bacitracin, Heomycin and Polymyxin B and rebandaged with a Bioclusive dressing.
  • strips of skin 0.5 cm x 3 cm x 10 cm were dis ⁇ sected so that the wound bed was in the center of the strip.
  • a strip of normal skin was also excised. The strips were tested for tensile strength using an Instron mechanical tester.
  • Two quadrants of the wound bed includ ⁇ ing peripheral normal skin were removed for histology. Two quadrants of the wound were removed for analysis of hydroxyproline and DNA content.
  • Hydroxyproline was calculated as hydroxyproline per dry weight. Total DNA content of the other wound quadrant was determined by extraction with perchloric acid and reaction with diphenolamine as described by Dische and modified by Burton. DNA was calculated as micrograms DNA per gram dry weight. The values for hydroxyproline and DNA were combined in order to calculate the hydroxyproline per DNA. Hydroxyproline is an estimation of the collagen content of the wound. DNA is an estimation of the cellularity of the wound.
  • Trichrome Picrosirius Red Mason's Trichrome stained sec ⁇ tion were visualized using a microscope. Photographed Mason's Trichrome stains collagen, blue; muscle, red; keratin, yellow; and fibrin, red.
  • protease nexin enhanced angiogenesi ⁇ of the granulation bed.
  • protease nexin was examined for angiogenic activity using an assay technique of assaying the chorioallantoic membrane as described in Thompson et al. , Journal of Pathology, Vol. 145:27-37 (1985).
  • Protease nexin was soaked into a three dimensional collagen sponge at various concentrations using a total of 0.1 ml per sponge diameter, 0.5 cm. 1 ml thick.
  • the sponges containing the protease nexin were applied to the surface of the chorioallantoic membrane (CAM) of 10 day embryos. After 4 days, the CAMs were removed, fixed in formalin, examined by microscopy and quantitated by the method of Thompson et al cited supra.
  • tissue sections of the wound beds were oriented so that paraffin embedded tissues could be sectioned parallel to the skin surface. These sections were also stained with Mason's Trichrome stain. The number of vessels ap ⁇ pearing in the granulation tissue could be quantified.
  • protease nexin has been observed to have a number of biological functions involving cell movement and the promotion of neural outgrowth by alterations of endothelial cell mobility. It has also been observed to affect the degradation of extracellular matrix.
  • the results obtained with a single dose of protease nexin onto the granulation bed of full thickness dermal wounds on guinea pigs indicates that protease nexin has an affect of promoting wound healing as evidenced by an increase in wound strength and by an early increase in collagen synthesis as determined by hydroxyproline content of the granulation tissue. Histological analysis has showed that protease nexin affects the cellularity of the wound bed and appears to promote formation of new blood vessels.
  • protease nexin affects wound healing by affecting the synthesis of matrix macromolecules present in the granula ⁇ tion bed.
  • Hydroxyproline values were determined as described above in connection with Figure 3 and were corrected by the DNA content of the wound to obtain a hydroxyproline per DNA. Values for normal skin are derived from skin excised in the creation of the wound. Control values are derived healing the wound beds in the absence of protease nexin. Experimental values were derived from wound beds treated with a single dose of protease nexin.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Materials Engineering (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Dermatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Materials For Medical Uses (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Procédé de cicatrisation de plaies utilisant une composiiton contenant de la protéase nexine-I (PN-I). L'invention concerne également des compositions à base de PN-I dispersée uniformément dans un support pharmaceutiquement acceptable.
PCT/US1991/002161 1990-04-05 1991-04-01 Emploi de protease nexine-i induisant la cicatrisation de plaies WO1991015233A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP91907406A EP0556178B1 (fr) 1990-04-05 1991-04-01 Emploi de protease nexine-i induisant la cicatrisation de plaies
DE69129071T DE69129071T2 (de) 1990-04-05 1991-04-01 Benutzung von protease nexin-i zur wundheilung

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US505,442 1990-04-05
US07/505,442 US5112608A (en) 1987-03-13 1990-04-05 Use of protease nexin-I to mediate wound healing

Publications (1)

Publication Number Publication Date
WO1991015233A1 true WO1991015233A1 (fr) 1991-10-17

Family

ID=24010332

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/002161 WO1991015233A1 (fr) 1990-04-05 1991-04-01 Emploi de protease nexine-i induisant la cicatrisation de plaies

Country Status (9)

Country Link
US (1) US5112608A (fr)
EP (1) EP0556178B1 (fr)
JP (1) JP3150338B2 (fr)
AT (1) ATE163861T1 (fr)
AU (1) AU647120B2 (fr)
CA (1) CA2079783A1 (fr)
DE (1) DE69129071T2 (fr)
ES (1) ES2114887T3 (fr)
WO (1) WO1991015233A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0653214A1 (fr) * 1993-11-11 1995-05-17 YISSUM RESEARCH DEVELOPMENT COMPANY of the Hebrew University of Jerusalem Compositions antibacteriennes topiques
US5641483A (en) * 1995-06-07 1997-06-24 Beaulieu; Andre Wound healing formulations containing human plasma fibronectin
WO1997025994A1 (fr) * 1996-01-18 1997-07-24 Hansson Hans Arne Regulation du processus de guerison
US6251859B1 (en) 1995-06-07 2001-06-26 BEAULIEU ANDRé Deepithelialized skin diffusion cell system

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8602626D0 (en) * 1986-02-04 1986-03-12 Ciba Geigy Ag Neurite-promoting factor
US5326562A (en) * 1986-06-03 1994-07-05 Incyte Pharmaceuticals, Inc. Pharmaceutical dosage unit for treating inflammation comprising protease nexin-I
US5196196A (en) * 1986-06-03 1993-03-23 Incyte Pharmaceuticals, Inc. Use of protease nexin-I in wound dressings
US5278049A (en) * 1986-06-03 1994-01-11 Incyte Pharmaceuticals, Inc. Recombinant molecule encoding human protease nexin
US5206017A (en) * 1986-06-03 1993-04-27 Incyte Pharmaceuticals, Inc. Use of protease nexin-I as an antiinflammatory
US5766897A (en) * 1990-06-21 1998-06-16 Incyte Pharmaceuticals, Inc. Cysteine-pegylated proteins
US5756094A (en) * 1991-03-27 1998-05-26 Trustees Of The University Of Pennsylvania Methods for stimulating follicular growth
US5705178A (en) * 1991-05-31 1998-01-06 Gliatech, Inc. Methods and compositions based on inhibition of cell invasion and fibrosis by anionic polymers
US5605938A (en) * 1991-05-31 1997-02-25 Gliatech, Inc. Methods and compositions for inhibition of cell invasion and fibrosis using dextran sulfate
CA2216870C (fr) * 1995-04-20 2002-11-19 New York University Compositions et procedes de regulation de la croissance des cheveux
US5733884A (en) 1995-11-07 1998-03-31 Nestec Ltd. Enteral formulation designed for optimized wound healing
US20050208114A1 (en) * 1998-03-24 2005-09-22 Petito George D Composition and method for healing tissues
US6673623B1 (en) 2000-09-12 2004-01-06 Novocure, Inc. Methods and compositions that control lipid production
US7041787B2 (en) * 2000-12-29 2006-05-09 Kimberly-Clark Worldwide, Inc. Design and use of advanced zinc chelating peptides to regulate matrix metalloproteinases
US6600057B2 (en) 2000-12-29 2003-07-29 Kimberly-Clark Worldwide, Inc. Matrix metalloproteinase inhibitors
US20030119073A1 (en) * 2001-12-21 2003-06-26 Stephen Quirk Sensors and methods of detection for proteinase enzymes
EP1661578B1 (fr) * 2003-09-03 2018-03-14 Toshikazu Nakamura Agent curatif et preventif de l'ulcere de la peau contenant un hgf de recombinaison humain
EP2226382A1 (fr) 2009-03-03 2010-09-08 B.R.A.I.N. Biotechnology Research and Information Network AG Protéase pour le conditionnement des blessures et soin de la peau

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4265233A (en) * 1978-04-12 1981-05-05 Unitika Ltd. Material for wound healing
EP0233838A2 (fr) * 1986-02-04 1987-08-26 Incyte Pharmaceuticals, Inc. Facteur stimulant les neurites, et procédé de préparation
US4904469A (en) * 1986-02-27 1990-02-27 Rohm Gmbh Chemische Fabrik Therapeutic agents for enzymatic wound cleaning
US5006252A (en) * 1986-06-03 1991-04-09 Invitron Purified protease nexin

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991005566A1 (fr) * 1989-10-16 1991-05-02 Invitron Corporation Anticoagulant a base de nexine de protease i/sulfate de dextran

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4265233A (en) * 1978-04-12 1981-05-05 Unitika Ltd. Material for wound healing
EP0233838A2 (fr) * 1986-02-04 1987-08-26 Incyte Pharmaceuticals, Inc. Facteur stimulant les neurites, et procédé de préparation
US4904469A (en) * 1986-02-27 1990-02-27 Rohm Gmbh Chemische Fabrik Therapeutic agents for enzymatic wound cleaning
US5006252A (en) * 1986-06-03 1991-04-09 Invitron Purified protease nexin

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Nature, Volume 342, issued 30 November 1989, R. MEIER er al., "Induction of glia-derived nexin after lesion of a peripheral nerve", pages 548-550, see entire disclosure. *
Neuroscience, Volumen 18, Number 3, issued 1986, NEEDELS et al., "Induction of a neurite-promoting factor in rat brain following injury or deafferentation", pages 517-526, see Abstract No. 82085220. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0653214A1 (fr) * 1993-11-11 1995-05-17 YISSUM RESEARCH DEVELOPMENT COMPANY of the Hebrew University of Jerusalem Compositions antibacteriennes topiques
US5514657A (en) * 1993-11-11 1996-05-07 Yissum Research Development Company Of The Hebrew University Topical antibacterial preparation
US5641483A (en) * 1995-06-07 1997-06-24 Beaulieu; Andre Wound healing formulations containing human plasma fibronectin
US6251859B1 (en) 1995-06-07 2001-06-26 BEAULIEU ANDRé Deepithelialized skin diffusion cell system
WO1997025994A1 (fr) * 1996-01-18 1997-07-24 Hansson Hans Arne Regulation du processus de guerison
US6174855B1 (en) 1996-01-18 2001-01-16 Astrazeneca Ab Pharmaceutical compositions comprising thrombin inhibitors and their use in the control of wound healing processes

Also Published As

Publication number Publication date
DE69129071T2 (de) 1998-10-29
AU7680391A (en) 1991-10-30
CA2079783A1 (fr) 1991-10-06
EP0556178A4 (fr) 1993-04-27
JPH05508148A (ja) 1993-11-18
ES2114887T3 (es) 1998-06-16
ATE163861T1 (de) 1998-03-15
EP0556178B1 (fr) 1998-03-11
US5112608A (en) 1992-05-12
JP3150338B2 (ja) 2001-03-26
EP0556178A1 (fr) 1993-08-25
AU647120B2 (en) 1994-03-17
DE69129071D1 (de) 1998-04-16

Similar Documents

Publication Publication Date Title
AU647120B2 (en) Use of protease nexin-I to mediate wound healing
US5196196A (en) Use of protease nexin-I in wound dressings
CA2321177C (fr) Matrice extracellulaire a base de fibronectine de recombinaison utilisee dans la cicatrisation des blessures
Chen et al. Characterization of biologic properties of wound fluid collected during early stages of wound healing
Ghahary et al. Immunolocalization of TGF-β1 in human hypertrophic scar and normal dermal tissues
Rogalski et al. Human leukocyte elastase induces keratinocyte proliferation in vitro and in vivo
JP2004043507A (ja) 細胞外マトリックスの蓄積を防止するためのトランスフォーミング増殖因子βの阻害
US6841355B2 (en) Methods and compositions for wound healing
WO1995013828A1 (fr) Applications de calreticuline et d'autres proteines associees au hyaluronan, salutaires pour la guerison des lesions
US5155038A (en) Use of thrombospondin to promote wound healing
US6025150A (en) Methods and compositions for wound healing
GB2321191A (en) Peptides for use in wound treatment
CA2321933C (fr) Matrice extracellulaire a base de peptides de fibronectine utilisee dans la cicatrisation des blessures
JP2002526382A (ja) 皮膚創傷の治療へのプロテアーゼ阻害剤の使用
EP0858808B1 (fr) Peptides applicables au traitement de lésions
La Celle et al. Blood-borne fragments of fibronectin after thermal injury
JPH0782172A (ja) 創傷治療剤
US20040110686A1 (en) Methods and compositions for enhancing fibroblast migration
Ksander et al. Exogenous transforming growth factor‐β2 enhances connective tissue formation in transforming growth factor‐β1—deficient, healing‐impaired dermal wounds in mice
WO1999042132A1 (fr) Utilisation d'un anticorps monoclonal anti-cd44 pour acclerer la cicatrisation des blessures
Scott et al. 77-NUMBER zyxwvutsrqponmlkjih
EP1164992A1 (fr) Procedes et compositions permettant de renforcer la migration des fibroblastes
Bodnar et al. 057 Activation of CXCR 3 Inhibits Endothelial Tube Formation Through the Modulation of M‐Calpain

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 2079783

Country of ref document: CA

Ref document number: 1991907406

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1991907406

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 1991907406

Country of ref document: EP